Wet Beveling of Microinjection Needles Utilizing Constant Air Pressure for Feedback on Needle Opening

Summary

This protocol describes the assembly of a pneumatic system for the delivery of pressurized air to a needle during the process of needle beveling. The protocol further describes the beveling process for creating sharp microinjection needles and how to gauge the relative opening size of the needle.

Abstract

Microinjection needles are a critical tool in the delivery of genome modification reagents, CRISPR components (guide RNAs, Cas9 protein, and donor template), and transposon system components (plasmids and transposase mRNA) into developing insect embryos. Sharp microinjection needles are particularly important during the delivery of these modifying agents since they help minimize damage to the embryo being injected, thereby increasing the survival of these embryos as compared to injection with non-beveled needles. Further, the beveling of needles produces needles that are more consistent from needle to needle as compared to needles opened by randomly breaking the needle tip by brushing the tip against an object (side of a coverslip, the surface of the embryo to be injected, etc.). The process of wet beveling of microinjection needles with constant pressure air delivered to the needle allows the person beveling the needle to know when the needle is open (presence of bubbles) and also gives a relative indication of how large a needle opening has been created. The relative opening size in the needle can be determined by adjusting the air pressure delivered to the needle until an equilibrium is reached and bubbles stop flowing from the tip of the needle. The lower the pressure at which the equilibrium is reached, the larger the needle size; and conversely, the higher the pressure, the smaller the needle size.

Introduction

Insect genetic modification is a process originally developed in Drosophila by Rubin and Spradling, and over the years, this process has been modified to create genetic modifications in other species¹. The process relies on the precise delivery of modification components microinjected into embryos at a specific window of time and location within the developing embryo^2,3,4. Sharp microinjection needles are a critical tool in the process of genetic modification of some insects, such as mosquitoes^4,5

Protocol

NOTE: The protocol as described below uses the Sutter BV-10 microcapillary beveled. However, this protocol can be modified for use with any model microcapillary beveled.

1. Assembly of regulator, pressure gauge, and air supply tubing

1. Cut a section of urethane tubing for the connection from the air supply to the base of the regulator (R; Section 1, Figure 1). The length of this section will depend on the distance from the air supply to .......

Discussion

Genetic modification of mosquitoes relies on precise microinjection of the modification materials (plasmids, guide RNAs, or proteins) into pre-blastoderm embryos^3,4,5,6,7,8. Crucial to this process are sharp needles that easily pierce the embryo during injection^2,4. A.......

Materials

Name	Company	Catalog Number	Comments
1.0 mm O.D. microcapillaries	World Precision Instruments
Beveler pedestal oil	Sutter Instruments	008
Bicycle fender clip	VeloOrange	R-clip 4-pack	https://velo-orange.com/products/vo-r-clip-4-pack
Boom Stand Microscope	AmScope		AMScope 3.5X-90X Trinocular LED Boom Stand Stereo Microscope or equivalent
BV-10 Beveler	Sutter Instruments	BV-10
Diamond abrasive plate	Sutter Instruments	104F	Diamond abrasive plate - extra fine (0.2 µ to 1.0 µ tip sizes)
Gasket, Buna-N	Clippard Instrument Laboratory, Inc.	11761-2-pkg	Used to seal connection on T  or L connectors, if not already included with these pieces
Hose Clamp	Clippard Instrument Laboratory, Inc.	5000-2-pkg
Hose connector	Clippard Instrument Laboratory, Inc.	CT4-pkg	Need 5 hose connectors
Microinjection Needle Holder	World Precision Instruments	MPH3-10	Needle holder for 1mm outer diameter microcapillaries
P-2000	Sutter Instruments		Any needle puller
Photo-Flo 200 Solution	B&H Photo, Video and Audio	BH #KOPF200P  MFR #1464510	wetting agent
Pressure Gauge	Clippard Instrument Laboratory, Inc.	PG-100	0-100 psi gauge
Reference wick	Sutter Instruments	X050300
Reference wick holder	Sutter Instruments	M100019
Regulator	Clippard Instrument Laboratory, Inc.	01-Mar	Need #10-32 ports for connections
Rubber Packing Sheet 6 inx 6 in	Danco	Model # 59849
T fitting	Clippard Instrument Laboratory, Inc.	15002-2-pkg
Threaded Bar			Either a threaded rod or bar with threaded end. Threads must be 10-32.
Urethane tubing	Clippard Instrument Laboratory, Inc.	URH1-0804-BLT-050